This project involves the use of subzero temperatures to elucidate mechanisms of enzyme catalysis, in particular to obtain information concerning the dynamic processes occurring during catalysis. By initiating the reaction at a very low temperature (e.g., -100 degrees) in a fluid aqueous organic solvent system and utilizing techniques such as absorption, fluorescence and circular dichroism spectroscopy to monitor environment-sensitive groups in the enzyme and substrate it is possible to detect, accumulate and characterize intermediates in enzyme-catalyzed reactions. Intermediates formed in the reactions of lysozyme, beta-galactosidase and fire-fly luciferase with specific substrates will be studied in this manner. Conditions necessary to obtain enzyme-substrate intermediates, in crystalline form trapped at subzero temperatures, and suitable for x-ray crystallographic studies will be determined for a number of enzyme-substrate-cryosolvent systems.